/. Embryo/, exp. Morph. Vol. 24, 2, pp. 393-403, 1970
393
Printed in Great Britain
Facial formation in normal and cadmium-treated
golden hamsters
By JAMES E. MULVIHILL, 1 STEPHEN H. GAMM2
AND VERGIL H. FERM 3
From the Department of Oral Histopathoiogy and Periodontology, Harvard
School of Dental Medicine, Department of Orthodontics, Boston
University School of Graduate Dentistry, and the Department
of Anatomy-Cytology, Dartmouth Medical School
SUMMARY
In the golden hamster the critical days of palatal development occur between days 12 and 14
of gestation. During this period the shelves undergo transposition from vertical positions
alongside the tongue to horizontal positions above it, and undergo fusion with each other.
Palatal transposition, per se, probably takes place on or about day 12. Present data are insufficientto indicate in which direction this occurs. Although epithelial remnants arestill present
in the midline of the palate on day 13, they have completely disappeared by day 14, thus
signaling the completion of the fusion process. Preosteoblast areas are clearly visible on
day 12 but actual ossification is first seen on day 13.
Cadmium apparently has a marked deleterious affect on the head mesoderm of golden
hamsters, causing the production of numerous malformations, including unilateral and
bilateral cleft lips and palates. It is suggested that the clefts found in cadmium-treated
hamsters are due to a mesodermal deficiency rather than to a delay in shelf transposition.
Marked abnormalities in cartilage formation and delays in ossification are also described.
The mechanism of action of cadmium—whether directly on the differentiating embryonic
tissue, or indirectly through action on the maternal tissues—remains to be elucidated.
INTRODUCTION
Recent investigations have established the easily reproducible teratogenic
effects of cadmium on the embryonic facial structures of the golden hamster
(Ferm, 1967; Ferm & Carpenter, 1967, 1968; Ferm, 1969). Among the malformations produced were unilateral and bilateral cleft lips and palates and
complex facial fissures.
In our attempts to understand the nature of the palatal clefts produced by
cadmium, it became necessary to define clearly the normal events of palatal
1
Author's address: Department of Oral Histopathology and Periodontology, Harvard
School of Dental Medicine, 188 Longwood Avenue, Boston, Massachusetts 02115, U.S.A.
2
Author's address: Department of Orthodontics, Boston University School of Graduate
Dentistry, Boston, Massachusetts, U.S.A.
3
Author's address: Department of Anatomy-Cytology, Dartmouth Medical School,
Hanover, N.H., U.S.A.
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J. E. MULVIHILL, S. H. GAMM AND V. H. FERM
development in the golden hamster. Although detailed descriptions of palatal
formation exist for the mouse (Walker & Fraser, 1956; Larsson, 1961.) and the
rat (Coleman, 1965), previous papers on the development of the golden hamster
do not deal specifically enough with the events of palatal closure (Graves, 1945;
Boyer, 1953).
In this study the events of palatal closure in golden hamster embryos are
discussed as a basis of subsequent considerations of the interference by cadmium
with the normal events of palatal development. The morphology of these
cadmium-produced effects is also presented.
MATERIALS AND METHODS
Ten pregnant golden hamsters (Cricetus auratus) were divided into control (5)
and experimental (5) groups. The day following the evening of conception was
designated day 1 of gestation. On the eighth day of gestation five experimental
animals were injected intravenously with a solution of cadmium sulfate
(3CdSO4.8H2O) in the amount of 2 mg per kg, made up in distilled water. The
five control animals received no treatment. Animals from both groups were sacrificed on each of days 12, 13 and 14 of pregnancy. The numbers of control and
experimental embryos obtained at each time interval were as follows: day 12,
3 controls and 3 experimentals; day 13, 2 controls and 7 experimental; day 14,
ABBREVIATIONS ON FIGURES
cleft area
cleft palate
er epithelial remnants
Ipp lateral palatine processes
nc nasal cartilage
ca
CP
nasal septum
ossification areas
po preosteoblast areas
tc thyroglossal cleft
us
0
FIGURE 1
Frontal sections through three different antero-posterior levels of the palates of
normal (A, C, E) and cadmium-treated (B, D, F) 12-day-old hamster embryos. All
stained with H. and E. x 525.
(A) Primary palate region: the lateral palatine processes {Ipp) have assumed a
horizontal position and are completely united with the intervening nasal septum (ns).
No ossification is evident in the preosteoblast areas (po).
(B) Primary palate region: the cartilaginous nasal septum (nc) is bifurcated and
directed more in a horizontal than in a vertical direction, causing the nose to be
extremely foreshortened. A bilateral cleft palate (cp) exists with the nose deviating to
the side of the smaller cleft.
(C) Anterior hard palate region: the lateral palatine process is vertically directed
alongside the tongue.
(D) Anterior hard palate: the cartilaginous nasal septum is bifurcated and horizontally directed. The lateral palatine processes are redundant in nature.
(E) Soft-palate region: extreme posterior portions of the lateral palatine processes
lying horizontally above floor of mouth.
(F) Soft palate region: the palatal region appears normal but a thyroglossal cleft
(tc) exists at base of tongue.
Facial formation and cadmium
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J. E. MULVIHILL, S. H. GAMM AND V. H. FERM
1 control and 2 experimentals. The heads of the embryos from both normal and
experimental groups were fixed in Bouin's solution, processed according to
standard histologic procedures, and embedded in paraffin wax. Serial sections of
7 /.i thickness were obtained in the frontal plane. Comparative sections were
stained with either H. and E., Masson's trichome, or M tiller's colloidal ferric oxide.
RESULTS
For clarity of presentation, the palate is divided into the following areas,
proceeding antero-posteriorly: primary palate, anterior hard palate, mid-hard
palate, posterior hard palate and soft palate. The following descriptive results
were obtained.
J 2-day normal
In the region of the primary palate, the cartilaginous nasal septum and nasal
capsule are well demarcated. The lateral palatine processes in this area are
horizontal and have completely joined with the nasal septum (Fig. 1 A). In all
other sections of the palate the palatine shelves are directed in a vertical direction alongside the tongue (Fig. 1C); the only exception to this being in the
extreme posterior region where the tongue is actually part of the floor of the
mouth and the palatal shelves are lying horizontally above it (Fig. 1 E). Although
'preosteoblast' (osteoid) areas (Larsson, 1961) can be seen throughout the palate,
no sign of actual ossification is yet discernible.
FIGURE 2
Frontal sections through three different antero-posterior levels of the palate of
normal (A, C, E) and cadmium-treated (B, D, F) 13-day-old hamster embryos.
All stained with H. and E. x 52-5.
(A) Primary palate region: similar to day 12 except for increased size of structure.
Ossification (o) has begun.
(B) Primary palate region: the nasal cartilage (nc) is bifurcated and the nasal septum
(ns) is deviated away from the cleft areas (ca). Ossification appears to be delayed
(po).
(C) Anterior hard palate region: the lateral palatine processes are undergoing fusion.
Epithelial remnants (er) are evident in the midline. Areas of ossification are
present.
(D) Anterior hard palate region: the nasal cartilage (nc) is bifurcated and the
nasal septum (ns) is deviated away from the cleft area(ctf). The palatine process (Ipp)
on the side of the cleft is redundant in nature.
(E) Posterior hard palate region: fusion is taking place between the lateral palatine
processes (Ipp). Areas of ossification (o) are present.
(F) Posterior hard palate region: the lateral palatine processes (Ipp) are still separated,
a thyroglossal cleft exists and there are no evident areas of ossification.
Facial formation and cadmium
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J. E. MULVIHILL, S. H. GAMM AND V. H. FERM
12-day cadmium-treated
Areas of congenital malformations are most evident in the anterior portions
of the palate. In the areas of the primary palate (Fig. 1B) and anterior hard
palate (Fig. 1D), the nose is extremely foreshortened. The vertical portion of
the cartilaginous nasal septum is completely absent. A bilateral cleft exists in the
area of the primary median palatal triangle and the nasal septum deviates to the
side of the narrower cleft (Fig. IB). In the anterior hard palate the lateral
palatine processes are quite small, if not almost redundant (Fig. ID). In the
posterior region an abnormality which was found in several cadmium-treated
animals was a thyroglossal cleft (Fig. 1F). Except for a decreased amount of
cartilaginous nasal capsule in the mid-hard palate, the palate itself appears
normal from the mid-hard palate posteriorly (Fig. 1D, F).
13-day normal
The lateral palatine processes have already undergone transposition and are
united with each other in the midline (Fig. 2C). Epithelial remnants are seen in
all areas of the secondary palate except in the most posterior aspects of the soft
palate and in the region of the primary median palatal triangle (Fig. 2A).
Fusion has not yet occurred between the nasal septum and the secondary palate
(Fig. 2 C). Bone formation is present in all sections of the hard palate to about the
same degree, except in the mid-hard palate where ossification is further advanced
(Fig. 2A, C).
FIGURE 3
Frontal sections through 3 different antero-posterior levels of the palates of normal
(A, C, E) and cadmium-treated (B, D, F) 14-day-old hamster embryos. All stained
with H. and E. x 52-5.
(A) Primary palate region: similar to day 13 except for increased size of structure
and for advanced level of ossification (o).
(B) Primary palate region: the nasal cartilage (nc) is bifurcated and the nose is
foreshortened. A bilateral cleft palate (cp) exists as the lateral palatine processes
(Ipp) are joined neither to each other nor to the nasal septum (ns). Bone formation
appears retarded.
(C) Anterior hard palate region: the palate has now become fused with the nasal
septum. The level of ossification (o) has advanced from that of the previous day.
(D) Anterior hard palate region: there exists a bifurcated nasal cartilage (nc), a
foreshortened nasal septum (ns) and a bilateral cleft palate (cp). The lateral palatine
processes (Ipp) are approximating each other in the midline. Ossification (o) appears
retarded.
(E) Mid-hard palate region: bone formation (o) has extended up to, but does not include, the median raphe of the palate.
(F) Mid-hard palate region: despite the abnormal character of the nose, the palate
itself appears to be within normal limits. Ossification (o) somewhat retarded.
Facial formation and cadmium
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J. E. MULVIHILL, S. H. GAMM AND V. H. FERM
13-day cadmium-treated
Unlike the previous day, malformations can be seen throughout the length of
the palate. The nose is foreshortened and markedly deviates away from the cleft
side (Fig. 2B, D). The cartilaginous nasal septum is bifurcated throughout its
length (Fig. 2B, D). The lateral palatine process on the cleft side appears redundant throughout the length of the hard palate. The ability of the palatal
shelves to undergo transposition is apparently not affected, as they are all horizontally located above the tongue (Fig. 2B, D, F). Bone formation is present
but markedly retarded when compared to normal (Fig. 2B, D, F).
14-day normal
The secondary palate is now fully formed (Fig. 3 A, C, E). Epithelial remnants
are no longer present in any areas of the palate indicating that fusion has been
completed (Fig. 3C, E). The nasal septum has completely fused with the anterior
portion of the hard palate and the vomerian bone beneath the cartilaginous
nasal septum is well calcified (Fig. 3C). Bone formation, in general, has progressed much farther and has extended to the midline of the palate in the midhard palate (Fig. 3E) and posterior hard palate.
14-day cadmium-treated
The animal exhibits a bilateral cleft which continues as a midline cleft partway into the hard palate. The nose once more is extremely blunted and has a
bifurcation of the cartilaginous nasal septum. In all areas where a cleft exists,
bone formation appears retarded. From the region of the mid-hard palate
posteriorly, however, where palatal formation appears normal, ossification also
appears normal (Fig. 3F). Despite the bilateral nature of the cleft the nasal
septum still appears to deviate to one side. In the region of the mid-hard palate,
it is of interest that, while the lateral palatine processes have undergone fusion
and appear quite normal, the nose itself exhibits a bifurcated septum (Fig. 3F).
DISCUSSION
In the golden hamster, formation of the secondary palate occurs between
days 12 and 14 of development. By day 12 the most anterior portions of the
lateral palatine processes have already united with the primary median palatal
triangle which is between them. As one proceeds posteriorly, the palatal shelves
slowly take on a more and more vertical inclination as they come to hang
alongside the developing tongue, except in the most posterior areas where the
tongue is actually part of the floor of the mouth and the palatine shelves are
horizontally directed above it.
Palatal transposition takes place on or about day 12 of gestation. This is in
disagreement with Boyer (1953), who reported that it occurred on day 11.
Facial formation and cadmium
401
Unfortunately, we have been unable to obtain sections from a palate undergoing transposition. Until we are able to do so, it is not possible to say whether
transposition takes place anteriorly-posteriorly as in the rat (Coleman, 1965), or
posteriorly-anteriorly as in the mouse (Walker & Fraser, 1956). By day 13 the
shelves have already undergone transposition. Indeed, the fusion process between
the shelves is almost complete. Although epithelial remnants are present in the
midline throughout the hard palate, there are none seen in the soft palate. This
is therefore consistent with Wood's hypothesis that the shelves in this area unite
with each other by mergence rather than by fusion (Wood & Draus, 1962).
By day 13 palatal bone formation in the normal hamster embryo appears to
have progressed farthest in the regions of the primary median palatal triangle
and the mid-posterior hard palate. These could be centers of ossification from
which bone formation proceeds both anteriorly and posteriorly.
When cadmium is administered to pregnant hamsters, marked effects are
noted in the facial development of the offspring. Among those noted in this paper
and in previous reports are unilateral and bilateral clefts of the palate, midline
clefts through the nose, thyroglossal clefts, and anophthalmia (Ferm, 1967;
Ferm & Carpenter, 1967, 1968; Ferm, 1969).
Cadmium appears to have marked deleterious effects on cartilage and bone
formation. In some animals the vertical cartilaginous nasal capsule was entirely
missing. In others it was greatly foreshortened and bifurcated. Bone formation
tended to be retarded in all areas where clefts were present. It is of interest that
in those areas where clefts were not present, bone formation appeared quite
normal. It is difficult to explain why a generalized bodily function, such as
ossification, should be so closely related to a local happening such as shelf
fusion; unless both processes represent two different expressions of derangement
of embryonic mesenchymal cell activity.
Several present observations, as well as those by previous authors, support
this view. The inability of the processes to join is most likely due to a mesodermal deficiency as suggested by Stark (Stark, 1961). The foreshortening of the
nasal process has already been mentioned. The bifurcation of the nasal
septum probably represents a lack of union between the medial nasal processes.
These processes are thought to merge with one another rather than fuse with
each other (Patten, 1961). Therefore, since these processes are always in contact
with each other, a midline cleft is most probably due to a mesodermal deficiency.
Similar reductions in tissue mass were also seen in the lateral palatine processes. Furthermore, there was no evidence that the shelves were not able to
undergo transposition at the proper time as has been reported following
cortisone administration (Walker & Fraser, 1956). Therefore, the wide clefts
were once again most probably due to a deficiency in mesodermal tissue, rather
than delayed shelf transposition.
Marin-Padilla has demonstrated the effect of the teratogens vitamin A and
dimethylsulfoxide on the hamster embryo (Marin-Padilla & Ferm, 1965;
402
J. E. MULVIHILL, S. H. GAMM AND V. H. FERM
Marin-Padilla, 1966#, b). Both of these agents have a marked effect on the embryonic mesenchyme. In a similar fashion, the administration of cadmium
sulfate may affect a vulnerable cephalic mesoderm and interfere with the role
this mesoderm plays during the morphogenetic mechanism of palatal closure.
The facial malformations thus produced could be a direct consequence of a
cadmium-induced mesodermal disturbance. The existence and nature of the
direct action of the cadmium on the embryonic mesenchyme have yet to be
determined.
The placental transfer of radioactive Cd109 has recently been demonstrated in
this experimental system (Ferm, Hanlon & Urban, 1969). Further studies are
needed to determine whether the mesodermal deficiency underlying the facial
deformities resulting from this treatment might be due to causes other than
direct embryonic damage. For example, these malformations may be due to
interference with placental transfer of some essential metabolite (Walker &
Fraser, 1956), or to the specific action of the teratogen on maternal enzymes and
metabolism, with secondary effects on the differentiating embryonic tissue.
RESUME
La formation de la face chez les Hamsters dores
normaux et traites au cadmium
Chez le Hamster dore, la periode critique de la formation du palais se place entre les
12eme et 14eme jours de la gestation. Pendant cette periode, les cretes se deplacent de la
position verticale sur les cotes de la langue a la position horizontale au dessus d'elle et se
fusionnent. La transposition du palais per se se place probablement au 12emejour, ou vers le
12eme jour. Les donnees actuelles sont insuffisantes pour indiquer dans quelle direction cela
se passe. Alors que des restes epitheliaux se retrouvent sur la ligne mediane du palais au jour 13,
ils ont completement disparu au jour 14, ce qui montre ainsi la completion du processus de
fusion. Des aires de preosteoblastes sont clairement visibles au jour 12 mais 1'ossification
reelle n'est perceptible qu'au jour 13.
Le cadmium exerce apparemment un effet deletere marque sur le mesoderme de la tete de
hamsters dores, provoquant la production de nombreuses malformations, y compris des
fentes labiales unilaterales ou bilaterales et des fentes palatines. II est suggere que les fentes
trouves dans les hamsters traites au cadmium sont dues a une deficience du mesoderme plutot
qu'a un retard de la transposition des cretes. Des anomalies severes de la formation du cartilage et de retards de 1'ossification ont egalement ete decrites.
Le mecanisme de Faction du cadmium, qu'elle soit directe sur le tissu embryonnaire en
differentiation, ou qu'elle agisse indirectement par l'intermediaire des tissus maternels, reste
a etre elucide.
We wish to acknowledge the technical assistance of Miss Lorraine Stevens and Miss Anna
Morse.
This investigation was supported by USPHS grants DE 02849 (J. M.), HD 026J 6 (V. F.),
and GM 10210 (V. F.).
Facial formation and cadmium
403
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